BBA - Bioenergetics (v.1554, #3)

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase by Peter R Rich; Stephen E.J Rigby; Peter Heathcote (137-146).
Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (PM, F and F) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F but when F is generated with hydrogen peroxide, is always accompanied by the narrow radical. When PM is produced at high pH with CO/O2, no EPR-detectable radicals are formed. Conversion of the CO/O2-generated PM into F when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F determined optically. It is concluded that low pH causes a change of protonation pattern in PM which induces partial electron redistribution and tryptophan cation radical formation in F. These protonation changes may mimic a key step of the proton translocation process.
Keywords: Cytochrome c oxidase; Hydrogen peroxide; Radical; EPR; ENDOR;

Changes in excitonic interactions of photosystem II (PSII) reaction centre (RC) pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor (pheophytin (Pheo)) were analysed using circular dichroism (CD). The CD spectrum of PSII RC shows positive bands at 417, 435 and 681 and negative bands at 447 and 664 nm. Oxidation of the primary donor by illuminating the sample in the presence of silicomolybdate resulted in nearly symmetric decrease of CD amplitudes at 664 and 684 nm. In the Soret region, the maximum bleaching of CD signal was detected at 449 and 440 nm. Accumulation of reduced Pheo in the presence of dithionite brought about much lower changes in CD amplitudes than P680 oxidation. In this case, only a small asymmetric bleaching at 680 and 668 nm in the red region and a bleaching at 445, 435 and 416 nm in the Soret region has been detected. Therefore, we suppose that the contribution of the Pheo of the primary acceptor to the total CD signal of RC is negligible. In contrast to the oxidation of primary donor, the light-induced change in the CD spectrum upon primary acceptor reduction was strongly temperature-dependent. The reversible CD bleaching was completely inhibited below 200 K, although the reduced Pheo was accumulated even at a temperature of 77 K. Since the temperature does not influence the excitonic interaction, the temperature dependence of the CD changes upon Pheo reduction does not support the model of Pheo excitonically interacting with the other chlorophylls (Chl) of the RC. We propose that Pheo should not be considered as a part of a multimer model.
Keywords: Photosynthesis; Chlorophyll; Pheophytin; Polarised spectroscopy;

Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF1) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF1 with Mg2+ produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k −2(ADP)=1.5×10−1 min−1 and k −2(ATP)≅10−3 min−1, respectively. As follows from the study, the noncatalytic sites of CF1 are not homogeneous. One of them retains the major part of endogenous ADP after CF1 precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.
Keywords: Chloroplast coupling factor 1 (CF1); ATP synthase; F1-ATPase; Noncatalytic site;

cAMP-induced modulation of the growth yield of Saccharomyces cerevisiae during respiratory and respiro-fermentative metabolism by Laurent Dejean; Bertrand Beauvoit; Ana-Paula Alonso; Odile Bunoust; Bernard Guérin; Michel Rigoulet (159-169).
The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast. By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways. Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content. In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP. (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins. This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield. (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield. It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism.
Keywords: cAMP; Mitochondria; Growth yield; Oxidative phosphorylation; Microcalorimetry; Saccharomyces cerevisiae;

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. by Adrián Reyes-Prieto; Mohammed El-Hafidi; Rafael Moreno-Sánchez; Diego González-Halphen (170-179).
The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, α-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.
Keywords: Alternative oxidase; Chlamydomonas; Cyanide adaptation; Cytochrome c oxidase; Polytomella;

Electric field effects on red chlorophylls, β-carotenes and P700 in cyanobacterial Photosystem I complexes by Raoul N. Frese; Miguel A. Palacios; Aissa Azzizi; Ivo H.M. van Stokkum; Jochen Kruip; Matthias Rögner; Navassard V. Karapetyan; Eberhard Schlodder; Rienk van Grondelle; Jan P. Dekker (180-191).
We have probed the absorption changes due to an externally applied electric field (Stark effect) of Photosystem I (PSI) core complexes from the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus elongatus and Spirulina platensis. The results reveal that the so-called C719 chlorophylls in S. elongatus and S. platensis are characterized by very large polarizability differences between the ground and electronically excited states (with Tr(Δα) values up to about 1000 Å3 f−2) and by moderately high change in permanent dipole moments (with average Δμ values between 2 and 3 D f−1). The C740 chlorophylls in S. platensis and, in particular, the C708 chlorophylls in all three species give rise to smaller Stark shifts, which are, however, still significantly larger than those found before for monomeric chlorophyll. The results confirm the hypothesis that these states originate from strongly coupled chlorophyll a molecules. The absorption and Stark spectra of the β-carotene molecules are almost identical in all complexes and suggest similar or slightly higher values for Tr(Δα) and Δμ than for those of β-carotene in solution. Oxidation of P700 did not significantly change the Stark response of the carotenes and the red antenna states C719 and C740, but revealed in all PSI complexes changes around 700–705 and 690–693 nm, which we attribute to the change in permanent dipole moments of reduced P700 and the chlorophylls responsible for the strong absorption band at 690 nm with oxidized P700, respectively.
Keywords: Synechocystis PCC 6803; Synechococcus elongatus; Spirulina platensis; Stark spectroscopy; Red chlorophyll; Photosystem I; Exciton interaction;

Properties of the Photosystem II (PSII) complex were examined in the wild-type (control) strain of the cyanobacterium Synechocystis PCC 6803 and its site-directed mutant D1-His252Leu in which the histidine residue 252 of the D1 polypeptide was replaced by leucine. This mutation caused a severe blockage of electron transfer between the PSII electron acceptors QA and QB and largely inhibited PSII oxygen evolving activity. Strong illumination induced formation of a D1–cytochrome b-559 adduct in isolated, detergent-solubilized thylakoid membranes from the control but not the mutant strain. The light-induced generation of the adduct was suppressed after prior modification of thylakoid proteins either with the histidine modifier platinum-terpyridine-chloride or with primary amino group modifiers. Anaerobic conditions and the presence of radical scavengers also inhibited the appearance of the adduct. The data suggest that the D1–cytochrome adduct is the product of a reaction between the oxidized residue His252 of the D1 polypeptide and the N-terminal amino group of the cytochrome α subunit. As the rate of the D1 degradation in the control and mutant strains is similar, formation of the adduct does not seem to represent a required intermediary step in the D1 degradation pathway.
Keywords: Cyanobacterium; Cytochrome b-559; D1 polypeptide; Photoinhibition; Photosystem II; Synechocystis PCC 6803;

Multiheme cytochromes c have been found in a number of sulfate- and metal ion-reducing bacteria. Geobacter sulfurreducens is one of a family of microorganisms that oxidize organic compounds, with Fe(III) oxide as the terminal electron acceptor. A triheme 9.6 kDa cytochrome c 7 from G. sulfurreducens is a part of the metal ion reduction pathway. We cloned the gene for cytochrome c 7 and expressed it in Escherichia coli together with the cytochrome c maturation gene cluster, ccmABCDEFGH, on a separate plasmid. We designed two constructs, with and without an N-terminal His-tag. The untagged version provided a good yield (up to 6 mg/l of aerobic culture) of the fully matured protein, with all three hemes attached, while the N-terminal His-tag appeared to be detrimental for proper heme incorporation. The recombinant protein (untagged) is properly folded, it has the same molecular weight and displays the same absorption spectra, both in reduced and in oxidized forms, as the protein isolated from G. sulfurreducens and it is capable of reducing metal ions in vitro. The shape parameters for the recombinant cytochrome c 7 determined by small angle X-ray scattering are in good agreement with the ones calculated from a homologous cytochrome c 7 of known structure.
Keywords: Cytochrome c; Cytochrome c 7; Cytochrome c maturation proteins; Heterologous expression; Geobacter sulfurreducens; Multiheme cytochrome c; Small angle X-ray scattering;

Author Index (212-213).